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BIOLOGICAL ACTIVITY OF MEDROGESTONE A NEW ORALLY ACTIVE PROGESTIN

C. REVESZ and C. I. CHAPPEL Ayerst Research Laboratories, Montreal, Canada [Received 12th February 1966)

Summary. A new synthetic orally active progestational agent 'Medro- gestone' was investigated and its activity compared to progesterone and medroxyprogesterone acetate (map). Medrogestone fulfils the criteria of a pure gestagen, it has a spectrum of biological activity similar to that of progesterone but unlike progesterone, it is orally active. Medro- gestone is free of undesirable side effects in experimental animals. It is being investigated clinically as an oral progestin (Carter, Faucher & Greenblatt, 1964).

INTRODUCTION The lack of oral effectiveness of progesterone inspired the search during recent years for progestational compounds which would be orally active. A number of such compounds have been synthesized, tested in animals and are now widely used in menstrual irregularities, habitual, threatened abortion, cycle regulation, treatment of infertility and/or in combination with oestrogens as contraceptives. However, many of these substances in addition to their pro¬ gestational activity produce certain side-effects such as androgenicity, masculinization of female foetuses and adrenal atrophy. The desirability of an orally active progestin which would be free from these objectionable properties led to the synthesis of a series of derivatives of 17-methylprogesterone (Deghenghi & Gaudry, 1961; Deghenghi, Revesz & Gaudry, 1963) and their testing as progestational agents. The biological activity of 6-methyl-6-dehydro-17- methylprogesterone, which appeared to be the most promising member of this series, forms the subject of this paper. It has the following structural formula: CH,

* Generic name of 6-methyl-6-dehydro-17-methylprogesterone. Trade name in U.S.A. is Colprone, also known as -62022. t Present address: Bio-Research Laboratories, Pointe-Claire, Quebec, Canada. 473

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 474 C. Revesz and C. I. Chappel METHODS Progestational activity McPhail's (1934) modification of the Clauberg test was employed using progesterone and medroxyprogesterone acetate (map) as reference compounds. Immature female rabbits were primed with a total dose of 15 ^g oestrone given over a period of 6 days. From the 7th to the 12th day of the experiment the test compound was given daily, subcutaneously (in 0-2 ml of sesame oil) or orally (in 2 ml of sesame oil). Twenty-four hours after the last dose was given the animals were killed and sections removed from the proximal, middle and distal portions of each uterine horn. Specimens were fixed in Bouin's solution, sectioned at 5 µ and stained with Harris's haematoxylin and eosin stain. The sections were evaluated according to McPhail's grading system. Duration of action ofprogestational activity The duration of effect of a single dose in the above test was determined by the method of Junkmann (1954). Progesterone, 17-hydroxyprogesterone caproate and map were used as reference compounds. Immature female rabbits were primed with oestrone as previously described. On the 7th day the compound was given in a single high dose subcutaneously. On the following days 0-025 ^g of oestrone was then given daily subcutaneously. Groups of five animals were killed 3, 7, 10, 13 and 17 days after receiving the test compound. Uterine sections were evaluated as above. Local progestational activity In this test McGinty, Anderson & McCullough's (1939) method was used with progesterone and map as reference compounds. Immature female rabbits were primed as described in the Clauberg test. On the 7th day of the experiment the rabbits were anaesthetized (Nembutal 30 mg/ kg i.v.) and the uteri were exposed. Ligatures were placed at each end of the upper 3 to 4 cm segment of each uterine horn. The upper ligatures were tied tightly and the lower ones left loose without disturbance of the blood supply to the tissue. The test compound (dissolved in 0-1 ml of sesame oil) was intro¬ duced into one of the isolated segments through an incision below the lower ligature. As the contents of the syringe were injected, the lower ligature was tightened. The same volume of sesame oil was injected into the other horn as a control. The rabbits were killed 72 hr later and sections of the isolated segment taken for histological examination.

Deciduoma test The method of Elton & Edgren (1958) was used, with progesterone and map as reference compounds. Female hooded rats of the Long-Evans strain (140 to 160 g) were ovariectomized, allowed to recover for a week and then primed daily, for 4 days, with 5 µg of oestrone subcutaneously. The test compound was then given subcutane¬ ously daily for the following 9 days. On the 5th day of treatment with the test compound, the left horn of the uterus was exposed and 2-0 mg of histamine

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 475 dihydrochloride (in 0-05 ml distilled water) was injected into the lumen, while the right horn served as control. The animals were killed the day after the last subcutaneous injection. The uteri were dissected and the right and left horns weighed separately. Maintenance ofpregnancy test in rats The effect of Medrogestone in maintaining pregnancy was determined according to the method described by Madjerek, de Visser, van der Vies & Overbeek (1960), with progesterone and map as reference compounds. Effect of multiple doses. Groups of six female hooded rats of the Long-Evans strain were placed in cages which contained two mature males per cage. Vaginal smears were taken daily. The day spermatozoa were found in the smears was counted as Day 1 of the pregnancy and the animal was removed from the cage. On the 9th day of pregnancy the rats were ovariectomized and the number of implantations present in the uteri recorded. The test compound was given subcutaneously once daily, or orally twice daily, from the 9th to the 20th day of pregnancy. On the 21st day of pregnancy the uteri were removed through an abdominal incision and the living foetuses counted and weighed. The number of dead foetuses was also recorded. Effect of a single dose. The procedure and reference compounds were the same as when multiple doses were given except that after ovariectomy on the 9th day of pregnancy a single dose of the test compound was administered sub¬ cutaneously in 0-5 ml of sesame oil. The number of live foetuses found in each animal, divided by the number of implantations at the time of ovariectomy, multiplied by 100, was used as an index of the ability of a compound to maintain pregnancy.

Ovulation inhibition in rats Ovulation was induced by the method of Wilson & Zarrow (1962). Nore¬ thynodrel, Enovid, Medrogestone and Medrogestone plus Premarin® were tested. Twenty-four-day-old female hooded rats were injected subcutaneously with 30 units of pmsg (pregnant mare serum gonadotrophin). Exactly 56 hr later, they were injected s.c. with 10 units of hcg (human chorionic gonadotrophin). The rats were killed and autopsied 24 hr after the hcg was given. The number of ova expelled from the oviduct was counted under the low power of a light microscope (Rowlands, 1942). The test compounds were given subcutaneously in 0-1 ml of sesame oil with the pmsg, 24 hr before the hcg and with the hcg. The ed50 for ovulation in¬ hibition was the daily dose of the test compound which reduced the number of ova by 50% as compared with the controls. Prevention ofpregnancy test Groups of five adult female hooded rats were injected with Medrogestone for 1 week before they were caged with sexually mature males. Treatment of the females was continued for 2 weeks after which the males were removed from the cages. Vaginal smears were taken during the experiment in order to

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 476 C. Revesz and C. I. Chappel detect spermatozoa. The females were checked for pregnancy 1 week after the males were removed.

Gonadotrophin suppressant activity This activity was determined in parabiotic rats using the methods of Bunster & Meyer (1933) and Biddulph, Meyer & Grumbeck (1940). Medrogestone was tested alone and in combination with Premarin®. Oestrone was used as the standard. Pairs of 38- to 39-day-old hooded female litter-mates were united by surgical attachment of the lateral skin and abdominal muscles. One of the partners was spayed and given the test compound subcutaneously in sesame oil daily for 10 days. The animals were killed and autopsied on the morning of the 11th day of the experiment. Percentage ovarian inhibition was calculated according to Miyake (1961): 100 [V-C) [V-VJ ' where V average ovarian of the intact partner united with the — weights vehicle-injected spayed female; C = average ovarian weights of the intact partner united with the test compound injected spayed female; and Vt = average weights of the intact partner united with the vehicle- injected intact female. Androgenic and anabolic activity The Herschberger, Shipley & Meyer (1953) assay was used with testosterone propionate, progesterone and map as reference compounds. Twenty-one- to 23-day-old male rats (40 to 45 g) were castrated and given the test compound subcutaneously for 7 days, beginning on the day ofcastration. The animals were killed the day after the last injection and the weight of the levator ani, ventral prostate and seminal vesicles was recorded. Initial and final body weights were also determined. Anti-androgenie effects Some progestational agents have been reported to have strong anti-androgenic effects. The compounds were assayed for this activity in castrated rats as described by Lerner, Bianchi & Borman (1960). Progesterone and map were the reference compounds. Twenty-one- to 23-day-old male hooded rats were castrated. Beginning on the day of castration the test compound alone and in combination with testos¬ terone propionate (t.p.) was given subcutaneously daily for 7 days. The animals were killed the day after the last injection and the levator ani muscle, ventral prostate and the seminal vesicles were removed and weighed. Body weight increases were determined. The effect of Medrogestone on the sex organs of intact male rats was also determined.

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 477 Effect on the developing foetus The effect of Medrogestone on the genitalia and reproductive system of female offspring of mothers treated during pregnancy was determined as described by Revesz, Chappel & Gaudry (1960). Changes in the genitalia and reproductive system of male offspring were determined by the method of Hamada, Neuman & Junkmann (1963). Test compounds were administered subcutaneously (in sesame oil), from the 15th to the 20th day of pregnancy, to female hooded rats of the Long-Evans strain. The pups were removed by Caesarean section on the 22nd day and were placed with foster mothers. On the day of birth the anogenital distance was measured and the pups weighed. Animals which survived to the 20th day were autopsied and the reproductive system examined. Oestrogenic activity This assay was performed with oestrone as the reference compound in a modified Allen & Doisy (1923) method. Female hooded rats (150 g body weight) were ovariectomized. Three weeks later the rats were primed with oestrone subcutaneously (2 x 7-5 ^g) on two consecutive days. Vaginal smears were taken in the afternoon of the following day and in the morning of the next day. Rats with comified epithelial cells in the vaginal smears were given the test compound subcutaneously daily for 3 days, 1 week after priming. On the 4th-day morning and afternoon, and the 5th-day morning, vaginal smears were again taken. The ed50 is expressed as the total dose at which 50% of the animals had cornified epithelial cells in the vaginal smears.

Effect on endocrine glands Spayed female rats were treated orally or subcutaneously with the test com¬ pound for 14 days. The animals were killed on the 15th day of the experiment and the weight and histological appearance of the adrenals, pituitary, thyroid and thymus recorded. Progesterone and map were the reference compounds.

Effect on water and electrolyte excretion Both normal and saline-loaded adrenalectomized rats were tested according to Llaurado (1961). Anti-aldosterone properties were also examined. Glycogen deposition test Glycogen deposition in Medrogestone-treated animals was determined in fasted adrenalectomized rats as described by Olsen, Thayer & Kopp (1944). Single injections of the test compound were given to one group of animals 7 hr before the liver was removed. In a second group of animals consecutive in¬ jections were administered 24 and 7 hr before the removal of the liver. Hydro- cortisone alcohol was used as reference compound. Anti-inflammatory properties Cotton pellet granuloma test. A single dose of the test compound was applied to carragheenin-soaked pellet irritants which were implanted subcutaneously in

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 478 C. Revesz and C. I. Chappel adrenalectomized rats as described by Bush & Alexander (1960). Hydro¬ cortisone was used as the reference compound. Croton oil granuloma pouch test. Rats bearing a croton oil granuloma pouch were prepared by the method of Selye (1953), and the test compound was injected directly into the pouch (Robert & Nezamis, 1957). map and hydrocortisone were used as reference compounds. Table 1

progestational activity in oestrogen primed rabbits

No. of Mean McPhail's score Compound rabbits 0-005* 0-01 0-025 0-05 0-1 Medrogestone, + 1- oral + 2-7 + 3-1 + 3-8

map, + 1-5 oral + 3·5 + 4 Progesterone, + 3·3 s.c. + 4 Medrogestone, + 0-5 s.c. + 1-9 + 3-6

MAP, + 3-6 s.c. + 3-8 + 3-5

* AU doses in mg.

RESULTS Progestational activity The progestational activity of a daily oral dose of 0-01 to 0-1 mg of Medro¬ gestone and map were equal as shown in Table 1. With subcutaneous adminis¬ tration, Medrogestone was approximately four times as potent as progesterone but less potent than map. Duration of action of progestational activity As shown in Table 2, when 10 mg of Medrogestone was given there was activity 17 days after a single subcutaneous dose. The activity found at that time was similar to that found with the same dose of 17-hydroxyprogesterone caproate but less than with map. Progesterone did not exhibit long-acting properties at the 10 mg dose level. Local progestational activity At 10 pg dose level Medrogestone showed a marked local progestational effect similar to progesterone and map.

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 479 Deciduoma test The results in Table 3 indicate that a daily dose of 1 mg of Medrogestone given subcutaneously for 9 days had a stronger deciduomagenic effect than progesterone at 1 mg or map at 2 mg dose.

Maintenance ofpregnancy in rats Multiple doses of Medrogestone maintained pregnancy in ovariectomized rats. Better results were produced with 5 mg Medrogestone subcutaneously Table 2 duration of action of progestational activity

Single Mean McPhail's score Compound dose (mgj Route rabbit) 10 13 17

Progesterone 100 s.c. + 3 + 2 + 1 + 1 17cc-Hydroxyprogesterone caproate 10-0 s.c. + 4 + 4 + 2-1 + 1 + 2-3 Medrogestone 10-0 s.c. + 4 + 4 + 2-6 + 2 + 2 10-0 s.c. + 4 + 4 + 3 + 3

* Days after single dose. Table 3

deciduoma test in rats

No. of Daily dose Weight of the uterine horn (mg) Compound rats (mgjrat) Right (control) Left (treated) Sesame oil 10 0-2 ml 69-0±7-0 97-0 + 7-0 Progesterone 8 0-5 57-0+1-9 90 0 + 9-4 8 0-8 62-0±2-7 191-0 + 28-3 10 1-0 63-0 ±4-0 183 0 + 45-0 10 2-0 81-0±5-0 351-0±7-0 Medrogestone 9 10 65-0 + 5-0 924-0+112-0 MAP 10 2-0 90-0+1-9 465-0+70-0 than with the same dose of progesterone (Table 4). When 10 mg was given orally, Medrogestone was slightly more active than map. Single subcutaneous doses of 5 to 10 mg of Medrogestone and map given at the time of ovariectomy were equally effective in maintaining pregnancy (Table 5). Progesterone showed the same effect but only at extremely high doses.

Ovulation inhibition Medrogestone was effective in inhibiting ovulation in rats. The subcutaneous ed 50 of Medrogestone alone was found to be 1-5 mg (Tableó). The correspond¬ ing dose of norethynodrel was 3-1 mg. The addition of 17oc-ethinyl oestradiol- 3-methylether (mestranol) to norethynodrel decreased the ed50 with sub¬ cutaneous administration to 0-25 mg. When given orally norethynodrel was more

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 480 C. Revesz and C. L Chappel effective than Medrogestone, the respective ed50 doses being 2-8 and 4-8 mg. However, the addition of 10 /ig of Premarin decreased the oral ed50 of Medro¬ gestone to 2-1 mg and 20 µg of Premarin decreased the ed50 of Medrogestone to 1-2 mg. Table 4 maintenance of pregnancy test in ovariectomized rats with multiple doses

Daily No. of No. of rats No. of No. of No. of dose spayed holding implantations live foetuses dead %of to foetuses Compound (mgjrat) preg¬ pregnancy on the day at autopsy at autopsy surviving and nant term with sur¬ of spaying (21 days) (21 days) foetuses route rats viving foetuses

Sesame oil 0-2 ml s.c. 5 46 Progesterone 5-0 s.c. 4 42 7 2 17 10-0 s.c. 6 75 20 18 67 Medrogestone 1-0 s.c. 3 29 17 5 59 2-0 s.c. 9 84 46 7 54-7 5-0 s.c. 4 40 40 100 10-0 oral 10 124 32 25-8 10-0 oral 18 208 30 14-4

Table 5 maintenance of pregnancy test in ovariectomized rats with a single injection

live births Single dose s.c. No. of treated No. of Compound (mglrat) pregnant rats -xlOO No. of implantations at ovariectomy Controls Sesame oil 0-2 ml 0 Progesterone 50-0 0 100-0 48 1500 52 200-0 76 Medrogestone 5-0 25-6 7-5 22-7 10-0 60-8 12-0 84-3 5-0 20-4 7-5 12-2 100 80

Prevention ofpregnancy Medrogestone at daily subcutaneous doses of 1-0 and 0-5 mg prevented pregnancy in all the animals, while 0-25 mg daily prevented pregnancy in 80% of the animals. The combination of 0-25 mg Medrogestone with 4 µg Premarin prevented pregnancy in 100%. Premarin alone did not prevent pregnancy in the rat at this dose level. Gonadotrophin suppressant activity A combination of 2 and 5 mg of Medrogestone and 1 /tg Premarin given orally suppressed ovarian weight increase by nearly 100%. Premarin alone, at

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 481 this dose level had no effect (Table 7). Subcutaneously, at the dose levels tested, the addition of Premarin did not increase the effectiveness of Medro¬ gestone. Medrogestone alone, either s.c. or orally, had little gonadotrophin suppressant activity. Table 6

ovulation inhibition in rats

No. Mean No. of Compounds Dose of rats ovajrat

Sesame oil 0-1 ml s.c. 54-2 + 2 Norethynodrel 0-5 mg s.c. 44-6 ±2 1-0 s.c. mg 35-4±2 3-1 2 0 mg s.c. 29-4 + 4 4-0 mg s.c. 25-6 + 2

Sesame oil 0-1 ml s.c. 12 53-9+1 Enovid 0-125mgs.c. 5 39-4+1 0-25 mg s.c. 5 26-2 ±2 0-5 mg s.c. 5 15-2 + 2 0-25 mg 1 -0 mg s.c. 5 13-6 + 2 2-0 mg s.c. 5 10-0+1 Sesame oil 0-5 ml oral 50-2 + 4 Enovid 1 -0 mg oral 39-6±5 2-0 mg oral 29-4 + 5 2-8 mg 4-0 mg oral 22-0 + 0

Sesame oil 0-2 ml s.c. 21 42-3 + 0 Medrogestone 0-63 mg s.c. 17 30-5 + 2 1 -25 mg s.c. 17 22-8±l 1-5 mg 2-5 mg s.c. 17 14-7+1 Sesame oil 0-2 ml oral 20 42-0 + 0 Medrogestone 1 -25 mg oral 17 34-0 + 2 2-5 oral 17 26-7±2 mg 4-1 mg 5-0 mg oral 17 18-3+1 10-0 mg oral 5 11-8+1 Sesame oil 0-2 ml oral 44-3±3 Premarin 10-0 mg oral 39-2 ±3 1-25 mg oral 27-2 + 3 Medrogestone+ oral Premarin 2-5 mg 20-4±2 2-1 mg (10 ßg) 5-0 mg oral 12-4 + 2 Sesame oil 0-2 ml oral 47-0±3 Premarin 200 /ig oral 38-2+1 0-32 mg oral 35-2+1 Medrogestone+ 0-63 mg oral 28-6±3 Premarin (20 µg) 1 -25 mg oral 22-2+1 - 1 -2 mg 2-5 mg oral 15-4 + 2 5-0 mg oral 11-6+1 J

Androgenie and anabolic activity in immature male rats The results presented in Table 8 indicate that Medrogestone in daily s.c. or oral doses up to 5-0 mg did not produce any androgenic effect. The increase in prostate weight produced by progesterone was not found after treatment with Medrogestone. map given subcutaneously at daily doses of 0-5 and 2-0 mg showed marked androgenicity.

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 482 C. Rêves and C. L Chappel Anti-androgenic test In this test the ability of the test compounds to counteract the effect of 0-03 mg of testosterone propionate ( .p.) given concomitantly was determined. Table 9 summarizes the results for progesterone, Medrogestone and map. Progesterone at a dose level of 25 mg exhibited anti-androgenic properties.

Table 7

gonadotrophin suppressant activity

Daily No. Spayed Intact Ovaries Ovarian Compound dose of uterus uterus weight (mg) inhibition (%) pairs weight (mg) weight (mg)

Intact controls 0-2 ml s.c. 135-6 + 33-8 32-5 + 4-6 (sesame oil) Castrated controls 0-2 ml s.c. 71-1+ 9-2 206-7+11-8 141-1+22-5 (sesame oil)

Premarin 0-01 ßg s.c. 56 ±3-6 179-6+14-4 103 ±43-0 35 Premarin 0-2 µg s.c. 50-1 ±4-2 87 +17-8 21-5+ 3-6 108

Medrogestone 1-0 mg s.c. 59-2+ 2-8 131 ± 19-2 82-5 + 21-8 54 Medrogestone 2-0 mg s.c. 66 ±11-0 157-7+14-6 88 +25-0 51

+ 1 -0 s.c. Medrogestone mg 69-2 ± 4-2 254-2 + 32-5 118 ± 10-1 21 Premarin { 0-01 µg s.c. 2-0 s.c. Medrogestone + mg 52-4+ 5-5 147 +22-8 75 +13-9 61 Premarin { 0-01 ^g s.c. Medrogestone + 2-0 mg s.c. 79-8+ 5-3 104-4+19-8 14-4+ 1-4 114 Premarin { 0-2 ^g s.c. Premarin 1 ^g oral 72 ± 2- 182 ; 12-8 144 +30-4 0 Medrogestone 2-0 mg oral 53 + 6-2 174 +24-4 120-5±15-6 19 Medrogestone 5-0 mg oral 63 ± 10-4 197 +22-0 126 +24-4 14 + 2-0 oral Medrogestone mg 73 ±16-2 82 ± 19-8 40 ±130 93 Premarin { 1 ßg oral 5-0 oral Medrogestone + mg 79 ±3-4 163 + 3-8 49-5+ 7-4 84 Premarin { 1 //g oral

Medrogestone had marked anti-androgenic effects at daily s.c. doses of 0-5, 2-0 and 5-0 mg. The anti-androgenic effect was slightly less pronounced when Medrogestone was given orally at dose levels of 2-0 and 5-0 mg. map showed only slight anti-androgenic effects with a daily s.c. dose of 2-0 mg only on the prostatic weight. Table 10 shows the results in intact animals. At daily doses of 2-0 and 5-0 mg given subcutaneously there was a significant decrease in the weight of the ventral prostate, seminal vesicles and levator ani.

Effect on developingfoetuses In female offspring of mothers given daily subcutaneous doses up to 10 mg of Medrogestone during pregnancy no changes were observed at birth in the anogenital distance nor was any masculinizing effect detectable at 20 days of

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 483 age. In contrast, the anogenital distance was increased at birth and the external and internal genitalia were masculinized at 20 days of age in female rats from mothers treated daily during pregnancy with subcutaneous doses of 0-5 mg of map. No masculinization occurred at daily subcutaneous doses up to 100 mg of progesterone. In male offspring of mothers given up to 10 mg of Medrogestone during pregnancy no change in the anogenital distance was observed at birth. How¬ ever, as seen in Table 11, hypospadias and cryptorchidism were observed in male offspring at 20 days of age. This anti-masculinizing effect was observed when 10 and 20 mg daily doses were given to the mother, while at a 5 mg-dose level (25 mg/kg) the males of only one litter out of seven were affected. At the 2-5 mg-dose levels of Medrogestone no changes were observed.

Table 8

androgenic and anabolic activity

Ventral Seminal Average Compound No. of Daily dose prostate vesicles Levator ani body weight rats (mgjrat) (mg) (mg) (mg) increase (g)

Sesame oil 165 0-2 ml s.c 8-5 + 0-7 9-8 + 0-1 17-1 + 1-0 27-1 Testosterone 243 0 03 s.c. 43-9+1-9 50-3 + 2-6 24-4+1-3 28-9 propionate 12 0-5 s.c. 105-0±2-6 175-5±5-0 46-4±l-2 300 Progesterone 12 2-0 s.c. 35-3 + 2-1 17-0±0-9 15-2 + 0-6 27-6 10 5-0 s.c. 29-9 + 2-4 13-2 + 0-6 14-8± 1-3 27-0 10 25-0 s.c. 28-8 + 2-8 14-9± 1-8 14-0+1-6 24-0 Medrogestone 10 2-0 s.c. 8-4 + 0-5 10-0±0-7 15-0±2-8 21-7 9 5-0 s.c. 11-3 + 0-9 12-7 + 0-8 18-7±0-8 31-7 10 2 0 oral 10-3 + 0-5 10-7 + 0-5 15-2±0-7 22-1 10 5-0 oral 10-6 + 0-4 11-5 + 0-3 16-5±0-8 27-9 12 0-5 s.c. 18-4±l-0 20-8 + 0-8 22-6 + 0-6 30-0 12 2-0 s.c. 40-0+1-2 28-1 + 0-7 24-4 + 0-6 360

Oestrogenic activity Medrogestone showed no oestrogenic effect at doses of 5 mg. The ed50 of oestrone was 1 -6 µg in our test.

Effect on endocrine glands No change in organ weight or histological appearance occurred in ovari¬ ectomized rats given 2 mg of Medrogestone s.c. (Table 12). Progesterone decreased the adrenal and thymus weights at extremely high doses, while map produced the same changes at low doses. The histological changes in the adrenals found with map were the same as those described by Logothetopoulos, Sharma & Kraicer (1961).

Effect on water and electrolyte metabolism In saline-loaded adrenalectomized animals, Medrogestone in s.c. doses of 2-0 and 3-0 mg did not produce definite changes in water and electrolyte excretion. At a dose of 4-0 mg, however, it showed some tendency to reduce

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 484 C. Revesz and C. L Chappel urine volume and electrolyte output. In intact rats, repeated daily injections of 1-0 mg of Medrogestone caused a small but significant increase in the total sodium and potassium excretion.

Table 9 anti-androgenic test

No. of Dose Ventral Seminal Levator ani Compound rats (mgjday) prostate vesicles (mg) (mg) (mg)

Sesame oil 165 0-2 ml s.c. 8-5 + 0-7 9-8 + 0-7 17-1 + 1-0 27-1 243 0-03 43-9+1-9 50-3 + 2-6 24-4+1-3 28-9

T.P. + 10 0-03 s.c. 50-8 + 2-7 21-3 + 0-8 32-0 Progesterone 2-0 s.c. 44-6±l-7 T.P. + 10 0-03 s.c. 49-0 + 2-1 39-7+1-3 17-9±0-8 31-0 Progesterone 5-0 s.c. T.P. + 10 0-03 s.c. 29-0+1-2 16-7+1-2 14-7+1-8 27-0 Progesterone 25-0 s.c.

T.P. + 10 003 s.c. 20-8 + 2-3 14-8+1-0 20-3+1-6 33-6 Medrogestone 20 s.c. T.P. + 10 003 s.c 17-4+1-0 12-3 + 0-4 19-3+1-3 32-1 Medrogestone 5-0 s.c Controls 0-2 ml oral 7-8 + 0-4 9-6 + 0-7 12-5+1-6 21-3 t.p. + 0 03 s.c. Medrogestone 2-0 oral 26-9±2-4 21-9+1-4 15-6± 1-5 27-3 s.c. t.p. + 003 + 1-0 13-8+1-0 Medrogestone 5-0 oral 19-6+1-2 16-2 25-1 t.p. 10 003 s.c. + 42-9±2-2 31-4+1-0 310 MAP 0-5 s.c. 53-0±2-l T.P. 10 003 s.c. + 34-8+1-6 47-9+1-7 31-0 + 2-2 260 MAP 2-0 s.c.

Table 10 effect of 7 days' treatment in intact male rats

Dose s.c. No. Ventral Seminal Levator ani Body weight Compound (mgjday) of prostate vesicles (mg) increase rats (mg) (mg) (g) Sesame oil 0-2 ml 91-2 + 6-4 84-4 + 8-0 68-4±6-2 32 0 Medrogestone 2-0 61-0±6-0 41-6 + 3-7 34-8 + 3-4 28-4 5-0 59-9 + 8-0 33-8±2-6 42-1 + 4-0 26-6

In adrenalectomized saline-loaded rats given ¿//-aldosterone acetate, Medro¬ gestone showed some anti-aldosterone properties at the 2-0 mg dose level. Glycogen deposition test Medrogestone did not increase liver glycogenesis at total doses of 1-0 and 2-0 mg s.c.

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 485 Anti-inflammatory properties Cotton pellet granuloma test. In this test Medrogestone did not exhibit anti- inflammatory or thymolytic properties at doses of 0-5, 1-0 and 2-0 mg/pellet. Croton oil granuloma pouch assay. The capacity of Medrogestone to inhibit the Table 11 effect of medrogestone on offspring when administered during pregnancy

No. dose No. of males Daily Normal of s.c. litters (mgjrat) females Hypospadias and Normal Hypospadias Cryptorchidism cryptorchidism 20-0 3 20-0 4 20-0 4 200 7 200 4 20-0 3 20-0 6 20-0 6 1 37 14 20 10-0 6 2 2 10-0 6 3 1 100 3 5 10-0 2 2 3 100 2 2 6 10-0 6 3 1 10-0 7 3 1 10-0 5 3 4 37 18 23 5-0 6 6 5-0 7 6 5-0 12 7 5-0 4 2 5-0 1 2 5-0 4 2 5-0 7 1 41 26 2-5 3 2-5 9 2-5 8 2-5 7 2-5 5 2-5 6 38 formation of the inflammatory exúdate in this test was very low when com¬ pared with map or hydrocortisone.

DISCUSSION The results of these tests suggests that Medrogestone has a number of unique properties which have been confirmed in the clinic. Medrogestone meets the requirements for an orally active, purely progestational agent. It is active in

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access 486 C. Rêves and C. I. Chappel the Clauberg and McGinty tests, has a prolonged effect when given subcutan¬ eously and is able to maintain pregnancy. In all these tests it compares favour¬ ably with map. However, in terms of secondary effects, map and Medrogestone are quite different, map has androgenic effect in castrated male rats and causes masculinization of female foetuses, whereas Medrogestone is completely nega¬ tive in these tests. The masculinizing effect of map is shown when 0-5 mg is given daily to pregnant rats. This androgenic effect has only been demonstrated, however, in experimental animals and has not been reported in man. Medro¬ gestone is not androgenic, but has a marked anti-androgenic activity in the

Table 12

effect on endocrine glands

No. Body weight (g) Adrenals Daily Route Thymus Thyroids Pituitary Compound dose of rats Initial Final (mg) (mg) (mg) (mg)

Control— 0-2 ml 8 97-0 144-0 40-7 508-0 12-2 11-0 Sesame oil + 3-6 + 8 + 2-5 + 40 + 0-5 ±0-4

Medrogestone 0-5 mg 102-0 168-0 39-5 419-0 12-4 10-5 ±2-3 ±3 + 1-7 ±24 ±0-6 ±0-4 1 -0 mg 100-0 175-0 38-3 470-0 13-5 11-0 ±2-4 ±5 ±2-5 ±41 ±6-7 ±0-6 2-0 mg 100-0 1730 38-0 414-0 12-4 10-8 + 4-3 + 3-7 + 2-0 + 38 + 0-6 + 0-4

Progesterone 1-0 mg 10 89-0 157-0 44-0 557-0 13-3 10-3 ±2-3 ±3 ±3 ±38 ±0-4 ±0-3 15-0 mg 10 860 162-0 24-3 486-0 13-8 11-2 ±2 + 4 ±2 + 38 + 0-6 ±0-5

0-5 mg 10 106-0 172-0 20-0 3190 12-5 9-8 ±2 ±4 ±1-4 ±19 ±11 ±0-5 1-0 mg 10 81-2 126-0 13-8 266-3 12-0 8-7 ±1-6 + 4-3 + 3 + 24 ±0-7 + 0-5 castrated male rat test. This property is evident in its effect on male offspring when high doses (above 5 mg daily) are given to the mother during pregnancy. At a 5 mg daily dose it caused feminization of the male offspring in only one of seven litters. This anti-androgenic effect might prove clinically useful in cases where a suppression of increased endogenous androgens would be advantageous, i.e. benign prostatic hypertrophy, Stein-Leventhal syndrome, hirsutism, etc. map has a strong depressant effect on the adrenal gland, causing adrenal atrophy in ovariectomized female rats. In contrast, Medrogestone did not cause adrenal atrophy at the same dose levels. Clinical experience with map is relatively recent, considering the time re¬ quired to accumulate clinical evidence in the endocrine field. It is, therefore, difficult to assess with certainty the importance of the secondary effects of this compound. However, from a pharmacological view-point the freedom from secondary effects in Medrogestone-treated animals offers definite advantages.

Downloaded from Bioscientifica.com at 10/02/2021 12:28:16PM via free access Biological activity of Medrogestone 487 ACKNOWLEDGMENTS The authors wish to thank Dr J. Guy Rochefort for the anti-inflammatory tests, and Mrs Marie Wehner and Mr A. Legault for their technical assistance.

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